U.S. patent application number 10/479872 was filed with the patent office on 2004-11-04 for anti-ngf antibodies for the treatment of various disorders.
Invention is credited to Shelton, David l.
Application Number | 20040219144 10/479872 |
Document ID | / |
Family ID | 23133215 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040219144 |
Kind Code |
A1 |
Shelton, David l |
November 4, 2004 |
Anti-ngf antibodies for the treatment of various disorders
Abstract
The present invention relates generally to methods of using,
anti-NGF antibodies in the treatment of various NGF-related
disorders, including asthma, arthritis and psoriasis. The methods
are effective in treating these disorders in a patient without
having a significant adverse effect on the immune system of the
patient.
Inventors: |
Shelton, David l; (Oakland,
CA) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
23133215 |
Appl. No.: |
10/479872 |
Filed: |
June 10, 2004 |
PCT Filed: |
May 9, 2002 |
PCT NO: |
PCT/US02/15284 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60294392 |
May 30, 2001 |
|
|
|
Current U.S.
Class: |
424/131.1 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 11/06 20180101; C07K 16/241 20130101; A61P 29/00 20180101;
A61P 25/00 20180101; A61P 43/00 20180101; C07K 16/468 20130101;
C07K 2317/92 20130101; A61P 21/00 20180101; C07K 2317/54 20130101;
C07K 2317/626 20130101; A61P 13/10 20180101; C07K 2317/21 20130101;
A61P 17/06 20180101; A61P 31/22 20180101; A61P 39/02 20180101; C07K
2317/73 20130101; A61P 1/04 20180101; C07K 16/22 20130101; A61P
35/02 20180101; A61P 37/02 20180101; C07K 2317/24 20130101; A61K
39/3955 20130101; A61P 19/02 20180101; A61K 31/573 20130101; C07K
2317/622 20130101; A61K 2039/507 20130101; C07K 2317/31 20130101;
C07K 2317/76 20130101; A61K 2039/505 20130101; C07K 16/4291
20130101; A61P 11/04 20180101; A61P 17/02 20180101; C07K 2317/55
20130101; A61K 39/39566 20130101; A61K 39/3955 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/131.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A method of controlling an NGF-related disorder in a human
patient, comprising administering to said patient an effective
amount of an anti-human NGF (anti-hNGF) monoclonal antibody capable
of binding hNGF with an affinity in the nanomolar range, and
inhibiting the binding of hNGF to human TrkA (hTrkA) in vivo,
wherein said antibody has no significant adverse effect on the
immune system of said patient.
2. The method of claim 1 wherein the binding affinity of said
antibody to hNGF is about 0.10 to about 0.80 nM.
3. The method of claim 2 wherein the binding affinity of said
antibody to hNGF is about 0.15 to about 0.75 nM.
4. The method of claim 2 wherein the binding affinity of said
antibody to hNGF is about 0.18 to about 0.72 nM.
5. The method of claim 1 wherein said antibody binds essentially
the same hNGF epitope as an antibody selected from the group
consisting of MAb 911, MAb 912, and MAb 938.
6. The method of claim 5 wherein said antibody binds essentially
the same hNGF epitope as the antibody MAb 911.
7. The method of claim 1 wherein said antibody is also able to bind
murine NGF (muNGF).
8. The method of claim 1 wherein said antibody is an antibody
fragment.
9. The method of claim 8 wherein said antibody fragment is selected
from the group consisting of Fab, Fab', F(ab').sub.2, Fv fragments,
diabodies, single-chain antibody molecules, and multispecific
antibodies formed from antibody fragments.
10. The method of claim 9 wherein said single-chain antibody
molecule is a single-chain Fv (scFv) molecule.
11. The method of claim 1 wherein said antibody is chimeric.
12. The method of claim 1 wherein said antibody is humanized.
13. The method of claim 1 wherein said antibody is human.
14. The method of claim 1 wherein said antibody is a bispecific
antibody.
15. The method of claim 14 wherein said bispecific antibody has an
anti-IgE specificity.
16. The method of claim 1 wherein said NGF-related disorder is
other than a disorder associated with the effect of NGF on the
neuronal system.
17. The method of claim 16 wherein said NGF-related disorder is an
inflammatory condition.
18. The method of claim 17 wherein said inflammatory condition is
selected from the group consisting of asthma, multiple sclerosis,
arthritis, lupus erythematosus, and psoriasis.
19. The method of claim 18 wherein said condition is asthma.
20. The method of claim 18 wherein said condition is arthritis.
21. The method of claim 20 wherein said arthritis is rheumatoid
arthritis.
22. The method of claim 18 wherein said condition is psoriasis.
23. The method of claim 17 wherein said antibody is administered in
combination with another therapeutic agent for the treatment of
said inflammatory condition.
24. The method of claim 19 wherein said antibody is administered in
combination with a corticosteroid.
25. The method of claim 24 wherein said corticosteroid is
bectomethasone diproprionate (BDP).
26. The method of claim 19 wherein said antibody is administered in
combination with an anti-IgE antibody.
27. The method of claim 26 wherein said anti-IgE antibody is
rhuMAb-E25 or rhuMAb-E26.
28. The method of claim 21 wherein said antibody is administered in
combination with another therapeutic agent for the treatment of
rheumatoid arthritis.
29. The method of claim 28 wherein said other therapeutic agent is
an anti-TNF antibody or an antibody or immunoadhesin specifically
binding a TNF receptor.
30. A pharmaceutical composition comprising a chimeric, humanized
or human anti-human NGF (anti-hNGF) monoclonal antibody capable of
binding hNGF with an affinity in the nanomolar range, and
inhibiting the binding of hNGF to human TrkA (hTrkA) in vivo,
wherein said antibody has no significant adverse effect on the
immune system of a patient, in combination with a pharmaceutically
acceptable carrier.
31. The pharmaceutical composition of claim 30 wherein said
antibody is an antibody fragment.
32. The pharmaceutical composition of claim 31 wherein
said-antibody fragment is selected from the group consisting of
Fab, Fab', F(ab').sub.2, Fv fragments, diabodies, single-chain
antibody molecules, and multispecific antibodies formed from
antibody fragments.
33. The pharmaceutical composition of claim 30 wherein said
antibody is a bispecific antibody.
34. The pharmaceutical composition of claim 33 wherein said
bispecific antibody is capable of specific binding to native human
IgE,
35. The pharmaceutical composition of claim 33 wherein said
bispecific antibody is capable of specific binding to native human
TNF or a native human TNF receptor.
36. The pharmaceutical composition of claim 30 further comprising
another pharmaceutically active ingredient.
37. The pharmaceutical composition of claim 36 wherein said other
pharmaceutically active ingredient is suitable for the treatment of
an inflammatory condition.
38. The pharmaceutical composition of claim 37 wherein said
inflammatory condition is selected from the group consisting of
asthma, multiple sclerosis, arthritis, lupus erythematosus and
psoriasis.
39. The pharmaceutical composition of claim 38 wherein said
inflammatory condition is asthma.
40. The pharmaceutical composition of claim 38 wherein said
inflammatory condition is arthritis.
41. The pharmaceutical composition of claim 40 wherein said
arthritis is rheumatoid arthritis.
42. The pharmaceutical composition of claim 41 wherein said
inflammatory condition is psoriasis.
43. An article of manufacture comprising: a container; a
pharmaceutical composition of claim 30; and instructions for using
the composition of matter to control an NGF-related disorder in a
human patient.
44. The article of manufacture of claim 43 further comprising a
second pharmaceutically active ingredient.
45. The article of manufacture of claim 44 wherein said second
pharmaceutically active ingredient is suitable for the treatment of
an inflammatory condition.
46. The article of manufacture of claim 45 wherein said
inflammatory condition is selected from the group consisting of
asthma, multiple sclerosis, arthritis, lupus erythematosus and
psoriasis.
47. The article of manufacture of claim 43 further comprising a
second container with a composition contained therein, wherein the
composition comprises a second antibody which binds an NGF receptor
and blocks ligand activation
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to methods of using
anti-NGF antibodies in the treatment of various NGF-related
disorders, including asthma, arthritis and psoriasis. The methods
are effective in treating these disorders in a patient without
having a significant adverse effect on the immune system of the
patient.
[0003] 2. Description of the Related Art
Nerve Growth Factor (NGF)
[0004] Nerve growth factor (NGF) was the first neurotrophin to be
identified, and its role in the development and survival of both
peripheral and central neurons has been well characterized. NGF has
been shown to be a critical survival and maintenance factor in the
development of peripheral sympathetic and embryonic sensory neurons
and of basal forebrain cholinergic neurons (Smeyne et al., Nature
368:246-249 (1994); Crowley et al., Cell 76:1001-1011 (1994)). NGF
upregulates expression of neuropeptides in sensory neurons (Lindsay
and Harmer, Nature 337:362-364 (1989)) and its activity is mediated
through two different membrane-bound receptors. The TrkA tyrosine
kinase receptor mediates high affinity binding and the p75
receptor, which is structurally related to other members of the
tumor necrosis factor receptor family, mediates low affinity
binding (Chao et al., Science 232:518-521 (1986)).
[0005] In addition to its effects in the nervous system, NGF has
been increasingly implicated in processes outside of the nervous
system. For example, NGF has been shown to enhance vascular
permeability (Otten et al., Eur.J.Pharmacol. 106:199-201 (1984)),
enhance T- and B-cell immune responses (Otten et al., Proc. Natl.
Acad. Sci. U.S.A. 86:10059-10063 (1989)), induce lymphocyte
differentiation and mast cell proliferation and cause the release
of soluble biological signals from mast cells (Matsuda et al.,
Proc. Natl. Acad. Sci. U.S.A. 85:6508-6512 (1988); Pearce et al.,
J. Physiol. 372:379-393 (1986); Bischoff et al., Blood 79:2662-2669
(1992); Horigome et al., J. Biol. Chem. 268:14881-14887
(1993)).
[0006] NGF is produced by a number of cell types including mast
cells (Leon et al., Proc. Natl. Acad. Sci. U.S.A. 91:3739-3743
(1994)), B-lymphocytes (Torcia et al., Cell 85:345-356 (1996),
keratinocytes (Di Marco et al., J. Biol. Chem. 268:22838-22846))
and smooth muscle cells (Ueyama et al., J. Hypertens. 11:1061-1065
(1993)). NGF receptors have been found on a variety of cell types
outside of the nervous system. For example, TrkA has been found on
human monocytes, T- and B-lymphocytes and mast cells.
[0007] Consistent with a non-neuronal role for NGF, an association
between increased NGF levels and a variety of inflammatory
conditions has been observed in human patients as well as in
several animal models. These include systemic lupus erythematosus
(Bracci-Laudiero et al., Neuroreport 4:563-565 (1993)), multiple
sclerosis (Bracci-Laudiero et al., Neurosci. Lett. 147:9-12
(1992)), psoriasis (Raychaudhuri et al, Acta Derm.Venereol.
78:84-86 (1998)), arthritis (Falcini et al., Ann. Rheum. Dis.
55:745-748 (1996)) and asthma (Braun et al., Eur. J. Immunol.
28:3240-3251 (1998)). Chronic inflammatory conditions such as these
are a significant public health problem. For instance, it is
estimated that arthritis affects 37.9 million people in the United
States alone. Current therapies for treating these conditions are
severely limited. An understanding of the role NGF plays in these
diseases may provide new methods for treating them.
[0008] A correlation between stress and psoriasis has been
observed. Based on this correlation and the symmetry of the
cutaneous lesions that accompany the disease, a relationship with
the nervous system has been proposed (Raychaudhuri et al., Acta
Derm. Venaercol. 78:84-86 (1998)). In particular, neuropeptides
have been suggested to play a role in the pathogenesis of
psoriasis. Investigators have reported an increased number of
terminal cutaneous nerves along with upregulation of one or more of
the neuropeptides, such as substance P (SP), vasoactive intestinal
polypeptide (VIP) and CGRP. NGF plays a role in regulating
innervation in the skin and also is known to upregulate
neuropeptides, suggesting that increased NGF levels may be
responsible for the upregulation of neuropeptides and the increased
cutaneous innervation seen with psoriasis. In fact, increased
expression of NGF has been observed in psoriatic keratinocytes
(Raychaudhuri et al., Acta Derm. Venercol. 78:84-86 (1998)). It has
been suggested that while NGF normally serves as a survival factor
for keratinocytes, overexpression of NGF prevents normal cell
death, leading to psoriasis (Pincelli at al., J. Derm. Sci.
22:71-79 (2000)).
[0009] A number of studies have indicated that neuropeptides such
as substance P (SP) and biologically active compounds released from
mast cells, such as histamine, also play a role in both naturally
occurring arthritis in humans and experimentally induced arthritis
in animal models (see e.g. Levine, J., Science 226:547-549 (1984)).
NGF has been shown to affect mast cell degranulation (Bruni at al.,
FEBS Lett. 138:190.193 (1982)) and substance P release (Donnerer et
al., Neurosci. 49:693-698 (1992)), implicating it in the
pathogenesis of arthritis.
[0010] Consistently, an elevated level of NGF in peripheral tissues
is associated with both hyperalgesia and inflammation and has been
observed in a number of forms of arthritis. The synovium of
patients affected by rheumatoid arthritis expresses high levels of
NGF while in non-inflamed synovium NGF has been reported to be
undetectable (Aloe et al., Arch. Rheum. 35:351-355 (1992)). Similar
results were seen in rats with experimentally induced rheumatoid
arthritis (Aloe et al., Clin. Exp. Rheumatol. 10:203-204 (1992)),
Elevated levels of NGF have been reported in transgenic arthritic
mice along with an increase in the number of mast cells. (Aloe et
al., Int. J. Tissue Reactions-Exp. Clin. Aspects 15:139-143
(1993)). However, purified NGF injected into the joint synovium of
normal rats does not induce knee joint inflammation, suggesting
that NGF does not play a causative role in arthritis (Aloe at al.,
Growth Factors 9:149.155 (1993)).
[0011] High NGF levels have been associated with allergic
inflammation and it has been suggested that this is related to mast
cell degranulation (Bonini et al., Proc. Natl. Acad. Sci. U.S.A.
93:10955-10960 (1996)).
[0012] Elevated NGF levels are also observed in both allergic and
non-allergic asthma (Bonini et al., supra). Mast cells, eosinophils
and T-lymphocytes have all been proposed to play a role in this
inflammatory disease and the correlation between NGF serum levels
and total IgE antibody titers suggests that NGF contributes to the
inflammatory immune response. Allergen induced airway inflammation
has been associated with increased local production of NGF in both
mice and humans (Braun et al., Int. Arch. Allergy Immunol.
118:163-165 (1999)).
[0013] NGF has been shown to regulate the development of increased
airway hyperactive response, a hallmark of bronchial asthma (Braun
et al., Eur. J. Immunol. 28:3240-3251 (1998)). Indeed, in one
study, treatment of allergen-sensitize mice with anti-NGF antibody
prevented the development of airway hyperresponsiveness following
local allergen challenge (Braun et al., Int. Arch. Allergy Immunol.
118:163-165 (1999)).
[0014] Despite the promising results obtained in mice, reported
adverse effects of neutralizing anti-NGF antibodies on the immune
system have raised serious questions about the feasibility of using
anti-NGF antibodies as a therapeutic in the prevention or treatment
of asthma or other diseases or disorders in human patients . In
particular, Torcia at al., Cell 85:345-356 (1996) identified NGF as
an autocrine survival factor for memory B lymphocytes, and
demonstrated that in vivo administration of neutralizing anti-NGF
antibodies caused a depletion of memory B-cells and abolished
secondary antigen-specific immune responses in mice. Garaci et al.,
Proc. Natl. Acad. Sci. USA 96:14013-14018 (1999) reported that NGF
is an autocrine survival factor that rescues human
monocytes/macrophages from the cytopathic effect caused by HIV
infection. This report, along with the findings of Torcia et al,
supra would suggest that anti-NGF antibodies have the potential of
compromising the immune system of the subject treated.
SUMMARY OF THE INVENTION
[0015] The present invention is based on the unexpected finding
that in vivo administration of a therapeutically effective amount
of an anti-NGF monoclonal antibody (antibody 911) had no adverse
effect on the immune system in an experimental mouse model of
allergy. Accordingly, this and related antibodies hold great
promise in the treatment of NGF-associated disorders, including
asthma, in human patients.
[0016] In one aspect, the invention concerns a method of
controlling an NGF-related disorder in a human patient by
administering to the patient an effective amount of an anti-human
NGF (anti-hNGF) monoclonal antibody that is capable of binding hNGF
with an affinity in the nanomolar range, and inhibiting the binding
of hNGF to human TrkA (hTrkA) in viva, wherein the antibody has no
significant adverse effects on the immune system of the
patient.
[0017] In one embodiment the binding affinity of the antibody to
hNGF is preferably about 0.10 to about 0.80 nM, more preferably
about 0.15 to about 0.75 nM and even more preferably about 0.18 to
about 0.72 nM.
[0018] In another embodiment, the antibody binds essentially the
same hNGF epitope as an antibody selected from the group consisting
of MAb 911, MAb 912 and MAb 938, more preferably the same epitope
as MAb 911.
[0019] In yet another embodiment the antibody is able to cross
react with murine NGF (muNGF).
[0020] The antibody may also be an antibody fragment, preferably an
antibody fragment selected from the group consisting of Fab, Fab',
F(ab').sub.2, Fv fragments, diabodies, single chain antibody
molecules and multispecific antibodies formed from antibody
fragments, and more preferably a single-chain Fv (scFv)
molecule.
[0021] In another embodiment the antibody is chimeric. It may also
be humanized or human.
[0022] In yet another embodiment the antibody is bispecific. The
bispecific antibody may have an anti-IgE specificity.
[0023] The NGF-related disorder that is controlled is preferably
not associated with the effect of NGF on the neuronal system.
[0024] In one embodiment the NGF-related disorder is an
inflammatory condition, preferably selected from the group
consisting of asthma, arthritis, multiple sclerosis, lupus
erythematosus and psoriasis.
[0025] In a preferred embodiment the condition is asthma. In
another embodiment the condition is arthritis, preferably
rheumatoid arthritis. In yet another embodiment the condition is
psoriasis.
[0026] In yet a further embodiment, the antibody is administered in
combination with another therapeutic agent for the treatment of an
inflammatory condition. Thus the antibody may be administered in
combination with another therapeutic agent for the treatment of
asthma. In one embodiment the antibody is administered with a
corticosteroid, preferably beclomethsone diproprionate (BDP). In
another embodiment the antibody is administered with an anti-IgE
antibody, such as rhuMAb-E25 or rhuMAb-E26. For the treatment of
rheumatoid arthritis, the antibody may be administered in
combination with an anti-TNF antibody or an antibody or
immunoadhesin specifically binding a TNF receptor.
[0027] In another aspect, the invention concerns a pharmaceutical
composition comprising a chimeric, humanized or human anti-human
NGF monoclonal antibody capable of binding hNGF with an affinity in
the nanomolar range and inhibiting the binding of hNGF to human
TrkA in vivo, wherein the antibody has no significant adverse
effects on the immune system of a patient, in combination with a
pharmaceutically acceptable carrier. The antibody in the
pharmaceutical composition may be an antibody fragment, preferably
an antibody fragment selected from the group consisting of Fab,
Fab', F(ab').sub.2, Fv fragments, diabodies, single-chain antibody
molecules and multispecific antibodies formed from antibody
fragments.
[0028] In one embodiment the antibody is a bispecific antibody. The
bispecific antibody may be capable of specific binding to native
human IgE or native human TNF or a native human TNF receptor.
[0029] In another embodiment the pharmaceutical composition further
comprises another pharmaceutically active ingredient, such as an
ingredient suitable for the treatment of an inflammatory condition.
The inflammatory condition is preferably one selected from the
group consisting of asthma, multiple sclerosis, arthritis, lupus
erythematosus and psoriasis. In one embodiment the inflammatory
condition is asthma. In another embodiment the inflammatory
condition is arthritis, preferably rheumatoid arthritis. In yet
another embodiment the inflammatory condition is psoriasis.
[0030] In another aspect, the present invention relates to an
article of manufacture comprising a container, a pharmaceutical
composition comprising a chimeric, humanized or human anti-human
NGF monoclonal antibody capable of binding hNGF with an affinity in
the nanomolar range and inhibiting the binding of hNGF to human
TrkA in vivo, wherein the antibody has no significant adverse
effects on the immune system of a patient, in combination with a
pharmaceutically acceptable carrier, and instructions for using the
composition of matter to control an NGF-related disorder in a human
patient.
[0031] In one embodiment the article of manufacture comprises a
further pharmaceutically active ingredient, preferably suitable for
the treatment of an inflammatory condition. The inflammatory
condition is preferably selected from the group consisting of
asthma, multiple sclerosis, arthritis, lupus erythematosus and
psoriasis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 summarizes the ability of six anti-NGF MAbs to bind
to NGF/NT-3 chimeric mutants. The relative binding of each MAb to
the NGF/NT3 mutants is compared to the binding of wildtype hNGF:
(-), <10%; (+), 10-30%; (++), 30-60%; (+++), 60-100%. The
EC.sub.50 of each MAb for binding to hNGF is: MAb 908,
1.8.times.10.sup.-10 M; MAb911, 3.7.times.10.sup.-M; MAb 912,
1.8.times.10.sup.-10 M; MAb 938, 7.4.times.10.sup.-10 M; MAb 14.14,
5.9.times.10.sup.-10 M.
[0033] FIGS. 2A-F shows the binding of MAbs to wild type and mutant
hNGF. The average EC.sub.50 values for each mutant obtained in 2 to
5 independent ELISA runs were compared to EC.sub.50 values obtained
for wild type NGF binding. EC.sub.50 values were determined by
linear regression analysis (unweighted) using the Kaleidagraph
software program (Abelbeck Software). Mutants resulting in at least
a two-fold reduction in MAb binding are designated by striped bars,
and the contributing residues are labeled.
[0034] FIG. 2A shows binding of MAb 908 to wild type and mutant
NGF.
[0035] FIG. 2B shows binding of MAb 909 to wild type and mutant
NGF.
[0036] FIG. 2C shows binding of MAb 911 to wild type and mutant
NGF.
[0037] FIG. 2D shows binding of MAb 912 to wild type and mutant
NGF.
[0038] FIG. 2E shows binding of MAb 938 to wild type and mutant
NGF.
[0039] FIG. 2F shows binding of MAb 14.14 to wild type and mutant
NGF.
[0040] FIGS. 3A-F presents molecular models of MAb epitopes on NGF
for anti-NGF MAbs 908, 909, 911, 912, 938 and 14.14, respectively.
The light and medium gray designations differentiate each monomer
of NGF, and residues identified by ELISA that affect MAb binding
are shown in black. Each of the variable regions is labeled in FIG.
3A.
[0041] FIGS. 4A and B show immunoblot analysis of anti-NGF MAb
binding to non-reduced hNGF (A) and reduced hNGF (B). Molecular
weight markers are visible in the first lane. Buffer and a negative
control antibody are shown in lanes 2 and 3, respectively. Under
non-reducing conditions, purified hNGF ran as a triplet set of
bands, which appear to correspond to monomeric, dimeric and
partially processed dimeric hNGF.
[0042] FIG. 5 summarizes the MAb epitope mapping results.
[0043] FIG. 6 shows that anti-NGF MAbs inhibited binding of hNGF to
a TrkA-IgG receptor immunoadhesin.
[0044] FIG. 7 shows the ability of anti-NGF MAbs to inhibit binding
of hNGF to a p75-IgG immunoadhesin.
[0045] FIG. 8 shows the ability of anti-NGF MAbs to inhibit binding
of hNGF to the TrkA extracellular domain expressed on transfected
CHO cells. Inhibition of tyrosine phosphorylation was measured by
ELISA using an antiphophotyrosine MAb.
[0046] FIG. 9 shows the ability of anti-NGF MAbs to inhibit the
survival effects of hNGF on embryonic rat dorsal root ganglion
neurons. Maximum survival was based on the signal obtained with NGF
alone, and maximal inhibition determined by the signal obtained
with the addition of saturating concentrations of soluble
TrkA-IgG.
[0047] FIG. 10 demonstrates that treatment with anti-NGF MAb 911
non-significantly increases the immune response to ovalbumin.
[0048] FIG. 11 shows that following immunization with chicken
gamma-globulin, treatment with anti-NGF MAb 911 non-significantly
reduces the immune response.
[0049] FIG. 12 shows that treatment with anti-NGF MAb 911 blocks
NGF induced thermal hyperalgesia.
[0050] FIG. 13 shows that in sensitized C57/BL6 mice (SN),
treatment with anti-NGF MAb 911 inhibits airway hyperreactivity
following challenge with inhaled dust mite antigen.
[0051] FIG. 14 shows that in sensitized C57/BL6 mice (SN),
treatment with anti-NGF MAb decreases infiltration of white blood
cells, lymphocytes and eosinophils into BAL following challenge
with inhaled dust mite antigen.
[0052] FIG. 15 shows that while cellular infiltration into BAL is
decreased in anti-NGF MAb treated sensitized mice, the proportion
of eosinophils remains high.
[0053] FIG. 16 shows that treatment with anti-NGF MAb 911
attenuates the increase of IL-13 in BAL following antigen
challenge.
[0054] FIG. 17 demonstrates that despite its ability to decrease
the inflammatory response to allergen, treatment with anti NGF MAb
does not decrease the humoral immune response as measured by total
serum immunoglobulin titer to dust mite.
[0055] FIG. 18 shows that treatment with anti-NGF MAb also does not
decrease the total serum level of IgE.
[0056] FIG. 19 shows that treatment with NGF increases the levels
of CGRP in the trigeminal ganglion, while treatment with anti-NGF
MAb produces a decrease in CGRP levels.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Definitions
[0057] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. See,
e.g. Singleton et al., Dictionary of Microbiology and Molecular
Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994);
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold
Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). One skilled
in the art will recognize many methods and materials similar or
equivalent to those described herein, which could be used in the
practice of the present invention. Indeed, the present invention is
in no way limited to the methods and materials described. For
purposes of the present invention, the following terms are defined
below.
[0058] As used herein, the terms "nerve growth factor" and "NGF"
are defined as all mammalian species of native sequence NGF,
including human. "NGF receptor" refers to a polypeptide that is
bound by or activated by NGF. NGF receptors include the TrkA
receptor and the p75 receptor of any mammalian species, including
humans.
[0059] The term "native sequence" in connection with NGF or any
other polypeptide refers to a polypeptide that has the same amino
acid sequence as a corresponding polypeptide derived from nature,
regardless of its mode of preparation. Such native sequence
polypeptide can be isolated from nature or can be produced by
recombinant and/or synthetic means or any combinations thereof. The
term "native sequence" specifically encompasses naturally occurring
truncated or secreted forms (e.g., an extracellular domain
sequence), naturally occurring variant forms (e.g., alternatively
spliced forms) and naturally-occurring allelic variants of the full
length polypeptides. "Antibodies" (Abs) and "immunoglobulins" (Igs)
are glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules which lack antigen specificity. Polypeptides of the
latter kind are, for example, produced at low levels by the lymph
system and at increased levels by myelomas. "Native antibodies and
immunoglobulins" are usually heterotetrameric glycoproteins of
about 150,000 daltons, composed of two identical light (L) chains
and two identical heavy (H) chains. Each light chain is linked to a
heavy chain by one covalent disulfide bond, while the number of
disulfide linkages varies between the heavy chains of different
immunoglobulin isotypes. Each heavy and light chain also has
regularly spaced intrachain disulfide bridges. Each heavy chain has
at one end a variable domain (VH) followed by a number of constant
domains. Each light chain has a variable domain at one end (VL) and
a constant domain at its other end; the constant domain of the
light chain is aligned with the first constant domain of the heavy
chain, and the light chain variable domain is aligned with the
variable domain of the heavy chain. Particular amino acid residues
are believed to form an interface between the light- and
heavy-chain variable domains (Chothia et al., J. Mol. Biol. 186:651
[1985]; Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592
[1985]; Chothia et al., Nature 342: 877-883 [1989]).
[0060] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
complementarity-determining regions (CDRs) or hypervariable regions
both in the light-chain and the heavy-chain variable domains. The
more highly conserved portions of variable domains are called the
framework (FR). The variable domains of native heavy and light
chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together in
close proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al. (1991) supra). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody-dependent cellular toxicity.
[0061] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0062] "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and -binding site. In a two-chain Fv
species, this region consists of a dimer of one heavy- and one
light-chain variable domain in tight, non-covalent association. In
a single-chain Fv species, one heavy- and one light-chain variable
domain can be covalently linked by a flexible peptide linker such
that the light and heavy chains can associate in a "dimeric"
structure analogous to that in a two-chain Fv species. It is in
this configuration that the three CDRs of each variable domain
interact to define an antigen-binding site on the surface of the
VH-VL dimer. Collectively, the six CDRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three CDRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0063] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0064] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called .kappa. and .lambda., based on the amino acid
sequences of their constant domains.
[0065] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these can be further divided into
subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1, and IgA.sub.2. The heavy-chain constant
domains that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon., .gamma., and .mu.
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0066] The term "antibody" specifically covers monoclonal
antibodies, including antibody fragment clones.
[0067] "Antibody fragments" comprise a portion of an intact
antibody, generally the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; single-chain antibody
molecules, including single-chain Fv (scFv) molecules; and
multispecific antibodies, such as bispecific antibodies, formed
from antibody fragments.
[0068] The term "monoclonal antibody" as used herein refers to an
antibody (or antibody fragment) obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly specific, being directed
against a single antigenic site. Furthermore, in contrast to
conventional (polyclonal) antibody preparations that typically
include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they are synthesized by the hybridoma culture, and are not
contaminated by other immunoglobulins. The modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any
particular method. For example, the monoclonal antibodies to be
used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567). The "monoclonal antibodies" also include
clones of antigen-recognition and binding-site containing antibody
fragments (Fv clones) isolated from phage antibody libraries using
the techniques described in Clackson et al., Nature, 352:624-628
(1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for
example.
[0069] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567 to Cabilly et
al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855
[19841]).
[0070] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a complementarity-determining region (CDR)
of the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are found neither
in the recipient antibody nor in the imported CDR or framework
sequences. These modifications are made to further refine and
optimize antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For further
details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et
al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol.,
596 (1992); and Clark, Immunol. Today 21: 397-402 (2000). The
humanized antibody includes a Primatized.TM. antibody wherein the
antigen-binding region of the antibody is derived from an antibody
produced by immunizing macaque monkeys with the antigen of
interest. "Single-chain Fv" or "scFv" antibody fragments comprise
the VH and VL domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally, the scFv
polypeptide further comprises a polypeptide linker between the VH
and VL domains, which enables the scFv to form the desired
structure for antigen binding. For a review of scFv see Pluckthun,
in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg
and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994),
Dall'Acqua and Carter, Curr. Opin. Struct. Biol. 8: 443-450 (1998),
and Hudson, Curr. Opin. Immunol. 11: 548-557 (1999).
[0071] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
931/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993).
[0072] An "isolated" antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0073] By "neutralizing antibody" is meant an antibody molecule
that is able to block or significantly reduce an effector function
of a target antigen to which it binds. Accordingly, a
"neutralizing" anti-NGF antibody is capable of blocking or
significantly reducing an effector function, such as receptor
binding and/or elicitation of a cellular response, of NGF.
"Significant" reduction means at least about 60%, preferably at
least about 70%, more preferably at least about 75%, even more
preferably at least about 80%; still more preferably at least about
85%, most preferably at least about 90% reduction of an effector
function of the target antigen (e.g. NGF).
[0074] An antibody is capable of "inhibiting the binding" of a
ligand to a receptor when it is capable of producing an objectively
measurable decrease in the ability of the ligand to bind the
receptor.
[0075] The term "epitope" is used to refer to binding sites for
(monoclonal or polyclonal) antibodies on protein antigens.
[0076] Antibodies which bind to a particular epitope can be
identified by "epitope mapping." There are many methods known in
the art for mapping and characterizing the location of epitopes on
proteins, including solving the crystal structure of an
antibody-antigen complex, competition assays, gene fragment
expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999. Competition assays are
discussed below. According to the gene fragment expression assays,
the open reading frame encoding the protein is fragmented either
randomly or by specific genetic constructions and the reactivity of
the expressed fragments of the protein with the antibody to be
tested is determined. The gene fragments may, for example, be
produced by PCR and then transcribed and translated into protein in
vitro, in the presence of radioactive amino acids. The binding of
the antibody to the radioactively labeled protein fragments is then
determined by immunoprecipitation and gel electrophoresis. Certain
epitopes can also be identified by using large libraries of random
peptide sequences displayed on the surface of phage particles
(phage libraries). Alternatively, a defined library of overlapping
peptide fragments can be tested for binding to the test antibody in
simple binding assays. The latter approach is suitable to define
linear epitopes of about 5 to 15 amino acids.
[0077] An antibody binds "essentially the same epitope" as a
reference antibody, when the two antibodies recognize identical or
sterically overlapping epitopes. The most widely used and rapid
methods for determining whether two epitopes bind to identical or
sterically overlapping epitopes are competition assays, which can
be configured in all number of different formats, using either
labeled antigen or labeled antibody. Usually, the antigen is
immobilized on a 96-well plate, and the ability of unlabeled
antibodies to block the binding of labeled antibodies is measured
using radioactive or enzyme labels.
[0078] The term amino acid or amino acid residue, as used herein,
refers to naturally occurring L amino acids or to D amino acids as
described further below with respect to variants. The commonly used
one- and three-letter abbreviations for amino acids are used herein
(Bruce Alberts et al., Molecular Biology of the Cell, Garland
Publishing, Inc., New York (3d ed. 1994)).
[0079] "Variants" are antibodies that differ in some respect from
native antibodies while retaining the same biological activity.
Variants may have an amino acid sequence that differs from the
sequence of the native antibody as a result of an insertion,
deletion, modification and/or substitution of one or more amino
acid residues within the native sequence. Variants may have a
different glycosylation pattern from native antibodies. Further,
variants may be native antibodies that have been covalently
modified.
[0080] A "disorder" is any condition that would benefit from
treatment according to the present invention. "Disorder" and
"condition" are used interchangeably herein and include chronic and
acute disorders or diseases, including those pathological
conditions which predispose the mammal to the disorder in question.
Non-limiting examples of disorders to be treated herein include
lupus erythematosus, contact dermititis, eczema, shingles,
postherpetic neuralgia, hyperalgesia, chronic pain, irritable bowel
disease, Crohn's disease, colitis, bladder cystitis, multiple
sclerosis, asthma, psoriasis, and arthritis, including chronic
arthritis and rheumatoid arthritis. A preferred disorder to be
treated in accordance with the present invention is an inflammatory
condition, such as asthma, multiple sclerosis, arthritis, lupus
erythematosus and psoriasis.
[0081] An "inflammatory condition" is a condition characterized by
one or more of pain, heat, redness, swelling and loss of function,
and is associated with tissue injury, infection, irritation or
damage.
[0082] The term "disease state" refers to a physiological state of
a cell or of a whole mammal in which an interruption, cessation, or
disorder of cellular or body functions systems, or organs has
occurred.
[0083] The term "effective amount or "therapeutically effective
amount" refers to an amount of a drug effective to treat and/or
prevent a disease, disorder or unwanted physiological condition in
a mammal. In the present invention, an "effective amount" of an
anti-NGF antibody may prevent, reduce, slow down or delay the onset
of a disorder such as lupus, multiple sclerosis, asthma, psoriasis
or arthritis; reduce, prevent or inhibit (i.e., slow to some extent
and preferably stop) the development of a disorder such as lupus,
multiple sclerosis, asthma, psoriasis or arthritis; and/or relieve,
to some extent, one or more of the symptoms associated with such a
disorder.
[0084] In the methods of the present invention, the term "control"
and grammatical variants thereof, are used to refer to the
prevention, partial or complete inhibition, reduction, delay or
slowing down of an unwanted event, e.g. physiological condition,
such as the inflammatory response associated with a disorder such
as asthma.
[0085] "Treatment" or "treat" refers to both therapeutic treatment
and prophylactic or preventative measures. Those in need of
treatment include those already with the disorder as well as those
prone to have the disorder or those in which the disorder is to be
prevented. For purposes of this invention, beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0086] A "significant adverse effect" on the immune system is an
effect that compromises the immune system and/or inhibits a normal
immune response to antigen challenge. An example of a significant
adverse effect on the immune system would be a reduced humoral
immune response.
[0087] "Pharmaceutically acceptable" carriers, excipients, or
stabilizers are ones which are nontoxic to the cell or mammal being
exposed thereto at the dosages and concentrations employed. Often
the physiologically acceptable carrier is an aqueous pH buffered
solution. Examples of physiologically acceptable carriers include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid; low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, arginine or lysine;-monosaccharides, disaccharides, and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN, polyethylene glycol (PEG), and
PLURONICS.
[0088] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as the anti-NGF antibodies disclosed
herein and, optionally, a chemotherapeutic agent) to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes.
[0089] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
[0090] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, cows, etc. Preferably, the mammal is human.
B. Methods for Carrying Out the Invention
[0091] As described in more detail below, administration of
anti-NGF monoclonal antibody 911 in a mouse model of asthma reduced
measures of airway hyperreactivity and inflammation but did not
decrease the humoral immune response to inhaled antigen as measured
by total serum immunoglobulin levels and serum level of IgE.
[0092] Anti-NGF antibodies are known in the art. The anti-NGF
antibodies useful in the present invention include polyclonal
antibodies, monoclonal antibodies, chimeric antibodies, humanized
antibodies, human antibodies, bispecific antibodies,
heteroconjugate antibodies, and antibody fragments, as well as
modified antibodies, including glycosylation variants of
antibodies, amino acid sequence variants of antibodies and
covalently modified antibodies. The antibodies can be made by any
method known in the art.
[0093] Thus, monoclonal antibodies may be made using the hybridoma
method first described by Kohler et al., Nature, 256:495 (1975), or
by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0094] Briefly, in the hybridoma method, a mouse or other
appropriate host animal, such as a hamster or macaque monkey, is
immunized to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the protein
used for immunization. Alternatively, lymphocytes may be immunized
in vitro. Lymphocytes then are fused with myeloma cells using a
suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice, pp.59-103, [Academic Press, 1986]). The hybridoma cells
thus prepared are seeded and grown in a suitable culture medium
that preferably contains one or more substances that inhibit the
growth or survival of the unfused, parental myeloma cells. For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine (HAT medium), which substances prevent
the growth of HGPRT-deficient cells. Preferred myeloma cell lines
are murine myeloma lines, such as those derived from MOP-21 and
MC.-11 mouse tumors available from the Salk Institute Cell
Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653
cells available from the American Type Culture Collection,
Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma
cell lines also have been described for the production of human
monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984);
Brodeur et al., Monoclonal Antibody Production Techniques and
Applications, pp. 51-63, Marcel Dekker, Inc., New York, [1987]).
Culture medium in which hybridoma cells are growing is assayed for
production of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies
produced by hybridoma cells is determined by immunoprecipitation or
by an in vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunosorbent assay (ELISA). The binding affinity of
the monoclonal antibody can, for example, be determined by the
Scatchard analysis of Munson et al., Anal. Biochem., 107:220
(1980). After hybridoma cells are identified that produce
antibodies of the desired specificity, affinity, and/or activity,
the cells may be subcloned by limiting dilution procedures and
grown by standard methods (Goding, Monoclonal Antibodies:
Principles and Practice, pp.59-103 (Academic Press, 1986)). The
monoclonal antibodies secreted by the subclones are suitably
separated from the culture medium, ascites fluid, or serum by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0095] Recombinant production of the antibodies requires the
isolation of DNA encoding the antibody or antibody chains. DNA
encoding the monoclonal antibodies is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences,
Morrison, et al., Proc. Nat. Acad. Sci. 81, 6851 (1984), or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
In that manner, "chimeric" or "hybrid" antibodies are prepared that
have the binding specificity of an anti-NGF monoclonal antibody
herein.
[0096] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody of the
invention, or they are substituted for the variable domains of one
antigen-combining site of an antibody of the invention to create a
chimeric bivalent antibody comprising one antigen-combining site
having specificity for a native NGF polypeptide and another
antigen-combining site having specificity for a different target,
such as a high-affinity IgE receptor, or a TNF receptor. Chimeric
or hybrid antibodies also may be prepared in vitro using known
methods in synthetic protein chemistry, including those involving
crosslinking agents. For example, immunotoxins may be constructed
using a disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate.
[0097] Non-human, such as murine antibodies can be humanized.
Generally, a humanized antibody has one or more amino acid residues
introduced into it from a non-human source. These non-human amino
acid residues are often referred to as "import" residues, which are
typically taken from an "import" variable domain. Humanization can
be essentially performed following the method of Winter and
co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the corresponding sequences of a human antibody.
[0098] It is important that antibodies be humanized with retention
of high affinity for the antigen and other favorable biological
properties. To achieve this goal, according to a preferred method,
humanized antibodies are prepared by a process of analysis of the
parental sequences and various conceptual humanized products using
three-dimensional models of the parental and humanized sequences.
Three dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e. the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the consensus and import sequence so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved. In general, the CDR residues are directly
and most substantially involved in influencing antigen binding. For
further details, see U.S. Pat. No. 5,821,337.
[0099] The invention also includes human anti-NGF antibodies. As
noted before, such human antibodies can be made by the hybridoma
method, using human myeloma or mouse-human heteromyeloma cell lines
for the production of human monoclonal antibodies (see, e.g.
Kozbor, J. Immunol. 133, 3001 (1984), and Brodeur, et al.,
Monoclonal Antibody Production Techniques and Applications,
pp.51-63 (Marcel Dekker, Inc., New York, (1987)). Furthermore, it
is possible to produce transgenic animals (e.g. mice) that are
capable, upon immunization, of producing a repertoire of human
antibodies in the absence of endogenous immunoglobulin production.
For example, it has been described that the homozygous deletion of
the antibody heavy chain joining region (J.sub.H) gene in chimeric
and germ-line mutant mice results in complete inhibition of
endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge. See,
e.g. Jakobovits et al., Proc. Natl. Acad. Sci. USA 90, 2551-255
(1993); Jakobovits et al, Nature 362, 255-258 (1993). For an
improved version of this technology, see also Mendez et al. (Nature
Genetics 15: 146-156 (1997)).
[0100] Alternatively, the phage display technology (McCafferty at
al., Nature 348, 552-553 (1990)) can be used to produce human
antibodies and antibody fragments in vitro, from immunoglobulin
variable (V) domain gene repertoires from unimmunized donors.
According to this technique, antibody V domain genes are cloned
in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, such as M13 or fd, and displayed as
functional antibody fragments on the surface of the phage particle.
Because the filamentous particle contains a single-stranded DNA
copy of the phage genome, selections based on the functional
properties of the antibody also result in selection of the gene
encoding the antibody exhibiting those properties. Thus, the phage
mimics some of the properties of the B-cell. Phage display can be
performed in a variety of formats; for their review see, e.g.
Johnson, Kevin S. and Chiswell, David J., Current Opinion in
Structural Biology 3, 564-571 (1993). Several sources of V-gene
segments can be used for phage display. Clackson at al., Nature
352, 624-628 (1991) isolated a diverse array of anti-oxazolone
antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes
from unimmunized human donors can be constructed and antibodies to
a diverse array of antigens (including self-antigens) can be
isolated essentially following the techniques described by Marks et
al., J. Mol. Biol. 222, 581-597 (1991), or Griffiths at al., EMBO
J. 12 725-734 (1993). In a natural immune response, antibody genes
accumulate mutations at a high rate (somatic hypermutation). Some
of the changes introduced will confer higher affinity, and B cells
displaying high-affinity surface immunoglobulin are preferentially
replicated and differentiated during subsequent antigen challenge.
This natural process-can be mimicked by employing the technique
known as "chain shuffling" (Marks et al., Bio/Technol. 10, 779-783
[1992]). In this method, the affinity of "primary" human antibodies
obtained by phage display can be improved by sequentially replacing
the heavy and light chain V region genes with repertoires of
naturally occurring variants (repertoires) of V domain genes
obtained from unimmunized donors. This technique allows the
production of antibodies and antibody fragments with affinities in
the nM range. A strategy for making very large phage antibody
repertoires (also known as "the mother-of-all libraries") has been
described by Waterhouse et al., Nucl. Acids Res. 21, 2265-2266
(1993), and the isolation of a high affinity human antibody
directly from such large phage library is reported by Griffiths et
al., EMBO J. 13: 3245-3260 (1994).
[0101] Gene shuffling can also be used to derive human antibodies
from rodent antibodies, where the human antibody has similar
affinities and specificities to the starting rodent antibody.
According to this method, which is also referred to as "epitope
imprinting", the heavy or light chain V domain gene of rodent
antibodies obtained by phage display technique is replaced with a
repertoire of human V domain genes, creating rodent-human chimeras.
Selection on antigen results in isolation of human variable domains
capable of restoring a functional antigen-binding site, i.e. the
epitope governs (imprints) the choice of partner. When the process
is repeated in order to replace the remaining rodent V domain, a
human antibody is obtained (see PCT patent application WO 93106213,
published 1 Apr. 1993). Unlike traditional humanization of rodent
antibodies by CDR grafting, this technique provides completely
human antibodies, which have no framework or CDR residues of rodent
origin.
[0102] The present invention specifically includes bispecific
antibodies. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy chain-light chain pairs, where the two heavy chains have
different specificities (Millstein and Cuello, Nature 305, 537-539
(1983)). According to a different and more preferred approach,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2 and CH3 regions. It is preferred to have the
first heavy chain constant region (CH1) containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance. For further details of generating bispecific
antibodies see, for example, Suresh at al., Methods in Enzymology
121, 210 (1986).
[0103] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection (PCT
application publication Nos. WO 91/00360 and WO 92/200373; EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0104] Antibody fragments have been traditionally derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et
al., Science 229:81 (1985)). However, these fragments can now be
produced directly by recombinant host cells. For example, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). In another embodiment, the
F(ab').sub.2 is formed using the leucine zipper GCN4 to promote
assembly of the F(ab').sub.2 molecule. According to another
approach, Fv, Fab or F(ab').sub.2 fragments can be isolated
directly from recombinant host cell culture. Other techniques for
the production of antibody fragments will be apparent to the
skilled practitioner.
[0105] For use in certain embodiments of the invention, it may be
desirable to modify the antibody fragment in order to increase its
serum half-life. This may be achieved, for example, by
incorporation of a salvage receptor binding epitope into the
antibody fragment (e.g., by mutation of the appropriate region in
the antibody fragment or by incorporating the epitope into a
peptide tag that is then fused to the antibody fragment at either
end or in the middle, e.g., by DNA or peptide synthesis). See WO
96132478 published Oct. 17, 1996.
[0106] The salvage receptor binding epitope generally constitutes a
region wherein any one or more amino acid residues from one or two
loops of a Fc domain are transferred to an analogous position of
the antibody fragment. Even more preferably, three or more residues
from one or two loops of the Fc domain are transferred. Still more
preferred, the epitope is taken from the CH2 domain of the Fc
region (e.g., of an IgG) and transferred to the CH1, CH3, or
V.sub.H region, or more than one such region, of the antibody.
Alternatively, the epitope is taken from the CH2 domain of the Fc
region and transferred to the C.sub.L region or V.sub.L region, or
both, of the antibody fragment.
[0107] Amino acid sequence variants, including substitution,
insertion and/or deletion variants, of the anti-NGF antibodies
specifically disclosed are prepared by introducing appropriate
nucleotide changes into the encoding DNA, or by peptide synthesis.
Methods for making such variants are Well known in the art, and
include, for example, "alanine scanning mutagenesis," as described
by Cunningham and Wells Science, 244:1081-1085 (1989). A particular
type of amino acid variant of an antibody alters the original
glycosylation pattern of the antibody. By altering is meant
deleting one or more carbohydrate moieties found in the antibody,
and/or adding one or more glycosylation sites that are not present
in the antibody.
[0108] Screening for Antibodies with the Desired Properties
[0109] Antibodies useful in the present invention are those that
neutralize the activity of NGF. Thus, for example, the neutralizing
anti-NGF antibodies of the present invention can be identified by
incubating a candidate antibody with NGF and monitoring binding and
neutralization of a biological activity of NGF. The binding assay
may be performed with purified NGF polypeptide(s), or with cells
naturally expressing, or transfected to express, NGF
polypeptide(s). In one embodiment, the binding assay is a
competitive binding assay, where the ability of a candidate
antibody to compete with a known anti-NGF antibody for NGF binding
is evaluated. The assay may be performed in various formats,
including the ELISA format.
[0110] The ability of a candidate antibody to neutralize a
biological activity of NGF can, for example, be carried out by
monitoring the ability of the candidate antibody to inhibit NGF
mediated survival in the embryonic rat dorsal root ganglia survival
bioassay as described in Hongo et al. (Hybridoma 19:215-227
(2000)).
[0111] To screen for antibodies which bind to an epitope on NGF
bound by an antibody of interest, a routine cross-blocking assay
such as that described in Antibodies, A Laboratory Manual, Cold
Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. Alternatively, or additionally, epitope mapping can be
performed by methods known in the art. For example, the NGF epitope
bound by a monoclonal antibody of the present invention can be
determined by competitive binding analysis as described in Fendly
et al. Cancer Research 50:1550 -1558 (1990). Cross-blocking studies
can be done by direct fluorescence on intact cells using the
PANDEX.TM. Screen Machine to quantitate fluorescence. In this
method the monoclonal antibody is conjugated with fluorescein
isothiocyanate (FITC), using established procedures (Wofsy et al.
Selected Methods in Cellular Immunology, p. 287, Mishel and Schiigi
(eds.) San Francisco: W.J. Freeman Co. (1980)). NGF expressing
cells in suspension and purified monoclonal antibodies are added to
the PANDEX.TM. plate wells and incubated, and fluorescence is
quantitated by the PANDEX.TM.. Monoclonal antibodies are considered
to share an epitope if each blocks binding of the other by 50% or
greater in comparison to an irrelevant monoclonal antibody
control.
[0112] Anti-NGF antibodies useful in the present invention can also
be identified using combinatorial libraries to screen for synthetic
antibody clones with the desired activity or activities. Such
methods are well known in the art. Briefly, synthetic antibody
clones are selected by screening phage libraries containing phage
that display various fragments (e.g. Fab, F(ab').sub.2, etc . . . )
of antibody variable region (Fv) fused to phage coat proteins. Such
phage libraries are panned by affinity chromatography against the
desired antigen. Clones expressing Fv fragments capable of binding
to the desired antigen are adsorbed to the antigen and thus
separated from the non-binding clones in the library. The binding
clones are then eluted from the antigen and can be further enriched
by additional cycles of antigen adsorption/elution. Suitable
anti-NGF antibodies for use in the present invention can be
obtained by designing a suitable antigen screening procedure to
select for the phage clone of interest, followed by construction of
a full length anti-NGF antibody clone by using the Fv sequences
from the phage clone of interest and a suitable constant region
(Fc) sequence.
[0113] The results obtained in the cell-based biological assays can
be followed by testing in animal, e.g. murine, models and human
clinical trials. If desired, murine monoclonal antibodies
identified as having the desired properties can be converted into
chimeric antibodies, or humanized by techniques well known in the
art, including the "gene conversion mutagenesis" strategy, as
described in U.S. Pat. No. 5,821,337.
C. Pharmaceutical Formulations
[0114] Therapeutic formulations of the antibody used in accordance
with the present invention are prepared for storage by mixing an
antibody having the desired degree of purity with optional
pharmaceutically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and may comprise buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN, PLURONICS or polyethylene
glycol (PEG).
[0115] The neutralizing anti-NGF antibodies useful in the methods
of the present invention may also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in
the art, such as described in Epstein et al., Proc. Natl. Acad.
Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA
77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Pat.
No. 5,013,556.
[0116] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst. 81(19):1484 (1989).
[0117] The active ingredients may also be entrapped in
microcapsuies prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0118] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0119] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably compounds with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide antibodies that bind to a different epitope of NGF
or to an NGF receptor in the one formulation. Alternatively, or
additionally, the composition may further comprise another
biologically active compound, such as an anti-inflammatory agent.
Such molecules are suitably present in combination in amounts that
are effective for the purpose intended.
[0120] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by, for example,
filtration through sterile filtration membranes.
[0121] Therapeutic anti-NGF antibody compositions are generally
placed into a container having a sterile access port, for example,
an intravenous solution bag or vial having a stopper pierceable by
a hypodermic injection needle.
D). Treatment with anti-NGF Antibodies
[0122] It is contemplated that, according to the present invention,
the anti-NGF antibodies may be used to treat various diseases or
disorders. Exemplary conditions or disorders include asthma,
psoriasis and arthritis. The anti-NGF antibodies may be used to
prevent the onset of the active disease state, to treat symptoms
that are currently being experienced and to treat the underlying
disease itself.
[0123] Despite advances in understanding the cellular and molecular
mechanisms that control allergic responses and improved therapies,
the incidence of allergic diseases, especially asthma, has
increased dramatically in recent years (Beasley et al., J. Allergy
Clin. Immunol. 105:466-472 (2000); Peat and Li, J. Allergy Clin.
Immunol. 103:1-10 (1999)). Allergic diseases can be treated, for
example, by allergen-based vaccination, in which increasing doses
of allergen are given by injection over years. Mild asthma can
usually be controlled in most patients by relatively low doses of
inhaled corticosteroids, while moderate asthma is usually managed
by the additional administration of inhaled long-acting
.beta.-antagonists or leukotriene inhibitors. However, the
treatment of severe asthma is still a serious medical problem.
Although an anti-IgE antibody currently awaiting FDA approval
(rhuMAb-E25, Xolair.TM., developed in collaboration of Genentech,
Inc., Tanox, Inc. and Novartis Pharmaceuticals Corporation) shows
promising results for early intervention in the treatment of
conditions that lead to symptoms of allergic asthma and seasonal
allergic rhinitis, there is need for the development of additional
therapeutic strategies and agents to control allergic diseases,
such as asthma.
[0124] The anti-NGF antibodies of the present invention can be used
for the treatment of asthma and other disorders associated with
airway hyperreactivity, typically characterized by episodes of
coughs, wheezing, chest tightness, and/or breathing problems.
[0125] The anti-NGF antibodies of the present invention are also
useful in the management of other inflammatory conditions, such as
multiple sclerosis, colitis, inflammatory bowel disease, bladder
cystitis, eczema, contact dermititis, arthritis, including chronic
arthritis nad rheumatoid arthritis, Crohn's disease, and
psoriasis.
[0126] In addition, anti-NGF antibodies are also useful in treating
other diseases that may be associated with increased levels of NGF
including, for example, lupus erythematosus, shingles, postherpetic
neuralgia, hyperalgesia, and chronic pain.
[0127] The anti-NGF antibodies are administered to a mammal,
preferably to a human patient, in accord with known methods, such
as intravenous administration, e.g., as a bolus or by continuous
infusion over a period of time, by intramuscular, intraperitoneal,
intracerebrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, or topical routes. Anti-NGF antibody can also be
administered by inhalation. Commercially available nebulizers for
liquid formulations, including jet nebulizers and ultrasonic
nebulizers are useful for administration. Liquid formulations can
be directly nebulized and lyophilized powder can be nebulized after
reconstitution. Alternatively, anti-NGF antibody can be aerosolized
using a fluorocarbon formulation and a metered dose inhaler, or
inhaled as a lyophilized and milled powder. For the treatment of
asthma and other conditions characterized by airway
hyperreactivity, a preferred route of administration is by
inhalation.
[0128] Other therapeutic regimens may be combined with the
administration of the anti-NGF antibody. The combined
administration includes co-administration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration, in any order, wherein preferably there
is a time period while both (or all) active agents simultaneously
exert their biological activities. For the treatment of asthma, it
might be particularly advantageous to use the antibodies herein in
combination with anti IgE antibodies, in particular rhuMAb-E25
(Xolair.TM.), or with second-generation antibody molecule
rhuMAb-E26 (Genentech, Inc.). The rhuMAb-E25 antibody is a
recombinant humanized anti-IgE monoclonal antibody that was
developed to interfere early in the allergic process. Combination
use also includes the possibility of administering the two
antibodies in a single pharmaceutical formulation, or using a
bispecific antibody, with anti-NGF and anti-IgE specificities. In
another preferred embodiment, the anti-NGF antibodies herein are
administered in combination with inhaled corticosteroids, such as
beclomethasone diproprionate (BDP) treatment. For the treatment of
rheumatoid arthritis or Crohn's disease, the antibodies of the
present invention can be administered in combination with other
treatment regimens known for the treatment of these conditions. For
example, the anti-NGF antibodies herein can be administered in
combination with Remicade.RTM. (Infliximab, Centocor), or
Enbrel.RTM. (Etanercept, Wyeth-Ayerst). The present invention also
includes bispecific antibodies targeting these diseases. For
example, a bispecific antibody could include an anti-TNF
specificity combined with the NGF-binding ability of the antibodies
herein.
[0129] Suitable dosages for any of the above co-administered agents
are those presently used and may be lowered due to the combined
action (synergy) of the agent and anti-NGF antibody.
[0130] For the prevention or treatment of disease, the appropriate
dosage of anti-NGF antibody will depend on the anti-NGF antibody
employed, the type of disease to be treated, the severity and
course of the disease, whether the antibody is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the antibody, and the discretion
of the attending physician. Typically the clinician will administer
the anti-NGF antibody until a dosage is reached that achieves the
desired result.
[0131] The anti-NGF antibody is suitably administered to the
patient at one time or over a series of treatments. Depending on
the type and severity of the disease, about 2 mg/kg of antibody is
an initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous or repeated dosing. A typical daily dosage might
range from about 1 g/kg to 100 mg/kg or more, depending on the
factors mentioned above. For repeated administrations over several
days or longer, depending on the condition, the treatment is
sustained until a desired suppression of disease symptoms occurs. A
preferred dosing regimen comprises administering an initial dose of
about 2 mg/kg, followed by a weekly maintenance dose of about 1
mg/kg of the anti-NGF antibody. However, other dosage regimens may
be useful, depending on the pattern of pharmacokinetic decay that
the practitioner wishes to achieve. The progress of this therapy is
easily monitored by conventional techniques and assays.
E. Articles of Manufacture
[0132] In another embodiment of the invention, an article of
manufacture containing materials useful for the treatment of the
disorders described above is provided. The article of manufacture
comprises a container and a label or package insert(s) on or
associated with the container. Suitable containers include, for
example, bottles, vials, syringes, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is effective for treating the
condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is an anti-NGF antibody. The
container may further comprise a second pharmaceutically active
agent. Preferably the second agent is suitable for the treatment of
an inflammatory disease such as asthma, multiple sclerosis,
arthritis, lupus erythematosus and psoriasis.
[0133] The label or package insert indicates that the composition
is used for treating the condition of choice, such as an
inflammatory condition. In one embodiment, the label or package
inserts indicates that the composition comprising the antibody that
binds NGF can be used to treat an inflammatory condition selected
from the group consisting of asthma, multiple sclerosis, arthritis,
lupus erythematosus and psoriasis. In addition, the label or
package insert may indicate that the patient to be treated is one
having asthma, psoriasis, arthritis or another disease or disorder.
Moreover, the article of manufacture may comprise (a) a first
container with a composition contained therein, wherein the
composition comprises a first antibody which binds NGF and inhibits
its biological activity; and (b) a second container with a
composition contained therein, wherein the composition comprises a
second antibody which binds an NGF receptor and blocks ligand
activation. The article of manufacture in this embodiment of the
invention may further comprise a package insert indicating that the
first and second compositions can be used to treat asthma,
psoriasis, arthritis or another disease or disorder. Alternatively,
or additionally, the article of manufacture may further comprise a
second (or third) container comprising a pharmaceutically
acceptable buffer, such as bacteriostatic water for injection
(BWFI), phosphate-buffered saline, Ringer's solution or dextrose
solution. It may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
[0134] Further details of the invention are -illustrated in the
following non-limiting examples.
EXAMPLES
[0135] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, VA.
Example 1
Production and Characterization of Anti-NGF Monoclonal
Antibodies
[0136] A. Production of anti-NGF Monolonal Antibodies
[0137] This example illustrates preparation of monoclonal
antibodies that can specifically bind human NGF (hNGF). Techniques
for producing monoclonal antibodies are well known in the art and
are described, for instance, in Kohler and Milstein, Nature
256:495-497 (1975). The experiments described in Examples 1 and 2
are further described in Hongo et al., Hybridoma 19:215.227.
[0138] A panel of 23 murine monoclonal antibodies to hNGF was
developed by a method analogous to that described in Hongo et al.,
Hybridoma 14:253-260. Briefly, Balb/c mice (Charles River
Laboratories, Wilmington, Del.) were immunized with human NGF in
Ribi adjuvant (Ribi Immunochem Research, Inc., Hamilton, Md.).
Splenocytes from the mouse demonstrating the highest titer of
antibody to immobilized NGF were fused with mouse myeloma cells
(X63.Ag8.653; American Type Culture Collection, Rockville, Md.).
After 10-14 days, supernatants were harvested and screened for
antibody production by enzyme-linked immunosorbent assay (ELISA).
Clones showing the highest immunoreactivity after the second round
of cloning were injected into Pristane-primed mice (Hoogenraad et
al., J. Immunol. Methods 6:317-320 (1983)) for in vivo production
of MAb. The ascites fluids were pooled and purified by affinity
chromatography (Pharmacia fast protein liquid chromatography;
Pharmacia, Uppsala, Sweden) using an established procedure (Moks et
al., Eur. J. Biochem. 85:1205-1210 (1986)) on staphylococcal
protein A (Pharmacia). The purified antibody preparations were
sterile filtered and stored at 4.degree. C. in phosphate-buffered
saline (PBS).
[0139] B. Epitope Mapping Using Domain Swap Mutants
[0140] Epitope specificity of anti-NGF MAbs was initially
determined by evaluating binding of the MAbs to chimeric
NGF/neurotrophin-3 (NT-3) proteins generated through
homolog-scanning mutagenesis. The use of such domain-swap mutants
has a distinct advantage over deletion mutants. The deletion of a
domain might disrupt the secondary structure of the protein whereas
substitution of a domain with a corresponding domain, of similar
size and substantially similar amino acid sequence, from a related
protein in domain-swap mutants is likely to retain the secondary
structure.
[0141] Eight hNGF/hNT-3 chimeric mutants, containing three to seven
residue substitutions of the human NT-3 (hNT-3) sequence into the
corresponding variable regions of hNGF (FIG. 1) were produced by
oligonucleotide-directed mutagenesis. The chimeric mutants were
transiently expressed in human 293 cells, and the binding of
anti-hNGF MAbs to mutant NGF was evaluated by enzyme-linked
immunosorbent assay (ELISA), as described below, using the purified
MAb as the capture antibody and an HRP-conjugated affinity-purified
rabbit anti-hNGF polyclonal antibody for detection. Binding of
anti-hNGF MAbs to each NGF mutant was determined from two to four
independent quantitative ELISA runs and was compared to binding to
wild-type NGF.
[0142] Briefly, microtiter plates (Nunc Maxisorb, VWR Scientific,
San Francisco, Calif.) were coated with 100 L per well of 1 g/mL
goat anti-mouse IgG (Boehringer-Mannheim, Indianapolis, Ind.)
overnight at 4.degree. C., washed, and the excess binding sites
were blocked with PBS containing 0.05% Tween 20 with 0.5% bovine
serum albumin (BSA, Intergen, San Diego, Calif.; PBS/BSA/T20). MAbs
(diluted to 1 g/mL in PBS/BSA/T20) were added to the appropriate
wells and incubated for 1-2 hours at ambient temperature. The
plates were washed, and 100 L of wild-type or mutant hNGF (diluted
in PBS/BSA/T20 to 60 ng/mL 7.8 ng/mL) were added, incubated for 1-2
hours at ambient temperature, and again washed. Purified rabbit
anti-hNGF polyclonal antibody conjugated to horseradish peroxidase
(HRP; 1:10,000 in PBS/BSA/T20) was added 1100 L/well) and incubated
for 1 hour. The plates were developed and read on dual wavelength.
Binding of the MAbs to the hNGF mutants was compared to wild-type
hNGF binding (set at 100%) analyzed under the same conditions.
[0143] Six human anti-NGF monoclonal antibodies (MAb 908, 909, 911,
912, 938 and 14.14) that demonstrated high affinities for hNGF
(EC.sub.50=0.18 nM for MAb 908, 0.18 nM for MAb 909, 0.37 nM for
MAb 911, 0.18 for MAb 912, 0.74 for MAb 938 and 0.59 for MAb 14.14)
and a greater than 60-90% reduction in binding to the various
chimeric mutants (FIG. 1) were selected for further analysis. As
shown in FIG. 1, three of the MAbs (908, 909 and 14.14) displayed
clear regional binding specificity, characterized by loss of 60-95%
maximal binding to a single variable region (FIG. 1). Less dramatic
effects of variable region mutants were observed for MAbs 911, 912
and 938, with multiple NGF variable regions contributing to the
binding epitopes of these antibodies. However, the variable regions
that comprise these epitopes are close together within the
three-dimensional structure (FIG. 3A).
[0144] C. Epitope Mapping Using Site-Directed Mutagenesis
[0145] To further define the epitope specificity of each of the six
anti-NGF MAbs selected, NGF mutants representing single, double, or
triple amino acid point mutations were generated, expressed and
characterized, with a particular focus on residues within regions
previously reported to play a role in TrkA and p75 binding and
biological function (Shih et al., J. Biol. Chem.,
269(44):27679-27686 (1994)). The effects of the mutations on
anti-NGF MAb binding were characterized by ELISA as described
above. Average EC.sub.50 values for binding of the anti-NGF MAbs to
each mutant were calculated from ELISA binding curves and compared
to EC.sub.50 values obtained for wild-type NGF binding (FIGS.
2A-F).
[0146] With all six MAbs, the binding specificity of the hNGF point
mutants generally correlated with the regions previously identified
by loss of binding to specific NGF chimeric mutants. However, the
effects of NGF point mutations on the binding of MAbs 911 and 938
were less prominent relative to the effects observed on the other
MAbs (FIGS. 2A-F). The regional specificity of the mutational
effects correlated with residues within or near variable regions
swapped in the NGF chimeric mutants, and which resulted in the loss
of more than 50-60% of the maximal binding of MAbs 911 or 938. For
MAb 911, these mutations included K32A+K34A+E35A, Y79A+T81K,
H84A+K88A, and R103A. Additional loss of binding was observed with
the nearby mutations E11A, Y52A and L112A+S113A (FIG. 2C). This
indicates that the epitope for MAb 911 spans four of the seven hNGF
variable regions (FIG. 3C).
[0147] D. Structural Dependency of Epitopes for Binding by MAbs
[0148] To determine whether binding of anti-NGF MAbs to hNGF
depends upon the structural conformation of epitopes on hNGF,
binding of the anti-NGF MAbs to non-reduced and reduced forms of
hNGF was evaluated. hNGF, either untreated or reduced by treatment
with mercaptoethanol, was subjected to gel electrophoresis and
transferred to nitrocellulose blots for immunoblotting. For
detection of hNGF, the nitrocellulose blots were washed, incubated
with primary and secondary antibodies, exposed to luminol substrate
(Amersham International, Amersham, U.K.) for 1 minute at ambient
temperature with agitation, and exposed to x-ray film (Eastman
Kodak, Rochester, N.Y.) for 10-45 seconds. In FIG. 4 it can be seen
that several monoclonal anti-NGF antibodies exhibit minimal binding
to reduced forms of hNGF. These included MAb 938, 908, 911 and 912,
indicating that epitopes on hNGF to which they bind are
structurally affected by charge alteration and reduction.
[0149] E. Molecular Modeling of Epitopes on anti-NGF Monoclonal
Antibodies
[0150] Molecular modeling representations of the identified
anti-NGF MAb epitopes (FIGS. 3A-F) were produced using the program
MidasPlus (University of California at San Francisco, San
Francisco, Calif.) and were based on coordinates for the
three-dimensional structure of murine NGF as previously described
(McDonald et al., Nature, 354:411-414(1991)).
[0151] A summary of the MAb epitope mapping results is presented in
FIG. 5.
Example 2
Determination of Neutralizing Activity of Anti-NGF Monoclonal
Antibodies
[0152] The observation that some epitopes mapped in regions
previously shown to be significant in the interaction of NGF with
TrkA and/or p75 suggested that the corresponding MAbs might also
block one or both of these interactions.
[0153] A. .sup.125I-hNGF Binding Assay.
[0154] To evaluate the possibility that one or more of the anti-NGF
MAbs might block NGF binding to TrkA and/or p75, the binding of
.sup.125I-hNGF to the TrkA-IgG receptor immunoadhesin was measured
in the presence of anti-NGF monoclonal antibodies.
[0155] Briefly, .sup.125I-hNGF was prepared using a modification of
the soluble lactoperoxidase method originally described by
Marchalonis (Marchalonis, Biochem J., 113:299-305 (1969)). The
final reaction mixture was fractionated over a pD-10 Sephadex G-25
size exclusion column (Pharmacia, Uppsala, Sweden) and stored at
4.degree. C. Microtiter plates (Nunc, Maxisorb) were coated
overnight at 4.degree. C. with purified rabbit anti-human IgG-Fc
specific polyclonal antibody (diluted to 2 g/ml in carbonate
buffer), washed with PBS, and blocked with 150 L of PBS/0.5% BSA
(PBS/BSA). Human TrkA-IgG or p75-IgG immunoadhesins (kindly
provided by Robert Pitti) (20 ng/mL) in PBS/BSA were added (100 L)
and incubated at ambient temperature for 1 hour. hNGF diluted in
PBS/BSA (150 pM final) was then added (100 L) and incubated for 1
hour at ambient temperature. hNGF (150 pM final) preincubated
overnight at 4.degree. C. with anti-NGF monoclonal antibodies (667
nM 0.58 nM) or an irrelevant monoclonal antibody not directed to
NGF was added in parallel and incubated as described. The plates
were washed with PBS containing 0.05% T20, and individual wells
were counted for 1 minute on a gamma counter (Packard Cobra Model
5010, Downers Grove, Ill.).
[0156] As can be seen FIG. 6, the anti-NGF monoclonal antibodies
inhibited binding of hNGF to the TrkA-IgG receptor immunoadhesin as
measured by the level of .sup.125I-labeled hNGF bound to TrkA-IgG.
All of the MAbs showed blocking ability at the highest
concentration assayed (667 nM) but clearly showed different
blocking capacity at lower concentrations. MAbs 911, 912 and 938
exhibited the same blocking potency (IC.sub.50.about.0.5-2 nM),
with 80-90% inhibition seen at 10 nM MAb or greater.
[0157] The ability of the MAbs to inhibit the binding of hNGF to
the low affinity p75 receptor was evaluated using p75-IgG in the
binding assay described above. As shown in FIG. 7, anti-hNGF MAbs
inhibit binding of hNGF to the p75-IgG receptor immunoadhesin using
.sup.125I-labeled hNGF. MAbs 911, 912 and 938 shows the highest
inhibitory activity with 75-90% reduction in binding observed in
the presence of less than 1 nM of MAb. MAb 909 also showed potent
blocking ability (>90% inhibition with 10 nM MAb), while MAbs
908 and 14.14 were significantly less potent (FIG. 7).
[0158] B. Kinase-induced Receptor Activation (KIRA) Assay.
[0159] A kinase-induced receptor activation (KIRA) assay was used
to measure NGF-dependent TrkA autophosphorylation in transfected
cells in response to stimulation with a ligand, such as hNGF,
and/or agonist monoclonal antibodies (Sadick at al., Exp. Cell Res.
234:354-361 (1997)). The anti-NGF MAbs were evaluated for their
ability to inhibit the binding of hNGF to the TrkA extracellular
domain expressed on CHO transfected cells and the subsequent
phosphorylation of tyrosine residues on TrkA (FIG. 8).
Phosphorylation of tyrosine residues was measured by ELISA using an
antiphosphotyrosine MAb.
[0160] Microtiter plates (Costar, Cambridge, Md.) were coated with
1.times.10.sup.5 chinese hamster ovary (CHO) cells expressing the
extra-cellular domain of the TrkA receptor with a Herpes simplex
virus glycoprotein D fragment (gD), a 26 amino acid polypeptide
serving as an epitope tag. Samples of either hNGF alone (150 pM)
(as a positive control) or hNGF (150 pM final) preincubated
overnight at 4.degree. C. with the individual anti-NGF MAbs (667 nM
0.31 nM final) were then added to wells containing the TrkA
expressing CHO cells (50 L per well) and incubated at 37.degree. C.
for 25 minutes. An irrelevant monoclonal antibody not directed
against NGF was used as a negative control. The hNGF-stimulated
cells were then treated with lysis buffer, and the lysates were
processed in gD-MAb-coated microtiter plates for the ELISA
detection of TrkA-containing phosphotyrosine similar to the
procedure described by Sadick et al (Sadick et al., Anal. Biochem.,
235:207-214 (1996)). 100 l of biotinylated 4G10 (monoclonal
anti-phosphotyrosine from Upstate Biologicals, Inc. (UBI, Lake
Placid, N.Y.)) diluted to 0.2 mg/ml in dilution buffer (PBS
containing 0.5% BSA, 0.05% Tween 20, 5 mM EDTA, and 0.01%
thimerosol) was added to each well. After incubation for 2 hours at
room temperature the plate was washed and 100 l HRP-conjugated
streptavidin (Zymed Laboratories, S. San Francisco, Calif.) diluted
1:50000 in dilution buffer was added to each well. The plate was
incubated for 30 minutes at room temperature with gentle agitation.
The free avidin conjugate was washed away and 100 l freshly
prepared substrate solution (tetramethyl benzidine, TMB,
two-component substrate kit, Kirkegard and Perry, Gaitehersbug,
Md.) was added to each well. The reaction was allowed to proceed
for 10 minutes, after which the color development was stopped by
the addition of 100 I/well of 1.0 M H.sub.3PO.sub.4. The absorbance
at 450 nm was read with a reference wavelength of 650 nm
(A.sub.450/460), using a Vmax plate reader (Molecular Devices, Palo
Alto, Calif.) controlled with a Macintosh Centris 650 (Apple
Computers, Cupertino, Calif.) and DeltaSoft software (BioMetallics,
Inc., Princeton, N.J.).
[0161] As shown in FIG. 8, all the selected anti-NGF monoclonal
antibodies could inhibit both the binding of hNGF to the TrkA
extracellular domain expressed on CHO transfected cells and
phosphorylation of tyrosine residues of the TrkA receptor.
Preincubation of hNGF with MAbs 911, 912 and 938 at the lowest
concentration tested (0.3 nM) resulted in approximately 60-80%
maximal inhibition of tyrosine phosphorylation relative to the
control MAb. MAbs 908, 909 and 14.14 were less potent.
[0162] C. Embryonic Rat Dorsal Root Ganglion Survival Bioassay.
[0163] Another assay used to determine the effects of anti-NGF
monoclonal antibodies on NGF-dependent processes was the embryonic
rat E14 dorsal root ganglia (DRG) survival bioassay. Anti-NGF
monoclonal antibodies were evaluated for their ability to inhibit
the survival effects of hNGF on embryonic rat E14 dorsal root
ganglia (DRG) neurons (FIG. 9).
[0164] Dorsal root ganglion (DRG) neurons obtained from E15 rats
(six to eight embryos) were cultured in F12 media with additives
(McMahon et al., Nat. Med. 8:774-780 (1995)) and 3 ng/mL of hNGF
either with or without anti-NGF monoclonal antibodies. After 72
hours of incubation at 37.degree. C., the cells were fixed with
formaldehyde and viable neurons were counted.
[0165] FIG. 9 shows the inhibition of DRG neuronal survival by
anti-NGF monoclonal antibodies. While MAbs 908, 909 and 14.14
reduced the survival by onle 20-30% at the highest concentration
(67 nM; 10 g/ml), MAbs 911 and 912 inhibited greater than 90%
survival at approximately 30-80 fold lower concentrations (0.8-2.4
nM; 0.12-0.37 g/ml). MAb 938 was able to inhibit survival activity
by 90%, but was 10-30 fold less potent than MAbs 911 and 912.
[0166] MAb 911 was the most potent blocker of the NGF/TrkA
interaction. MAb 911 recognizes an epitope containing the
overlapping NGF-TrkA and p75 binding region -turn A'-A" (V-1) and
the dominant TrkA binding region in-sheet (FIG. 30). The second
strongest NGF/TrkA inhibitor, MAb 912, recognizes the residues K32,
K34 and K35, a region previously shown to be crucial for the
NGF/p75 interaction. MAb 938, another potent blocker of TrkA and
p75 binding also recognizes regions critical for TrkA or p75
binding, the N- and C-termini.
[0167] The observed differences in antibody blocking specificity
are not due to the relative affinities of the MAbs toward hNGF
because two of the three most potent blocking antibodies (911 and
938) have lower affinities for NGF than the weaker blocking
MAbs.
[0168] NGF regions critical for binding to MAbs 911 and 912 overlap
with regions identified as critical regions for TrkA and p75
binding. MAbs 911 and 912 are capable blockers of NGF-induced
activities, indicating that these would be specific antagonists of
in vivo activities such as inflammatory hyperalgesia.
Example 3
The Effect of anti-NGF on the Immune Response
[0169] Mice were immunized subcutaneously with 10 g ovalbumin on
day 0 and allowed to recover. On day 40 following immunization
animals were injected IP with 10 mg/kg anti-NGF antibody 911 or a
control, isotype matched antibody. Two days later animals were
boosted with ovalbumin. The immune response was measured by ELISA
on serum samples taken on day 47. As can be seen in FIG. 10, there
was no significant (p>0.05) difference in the immune response
between the animals receiving anti-NGF antibody 911 and animals
receiving the control antibody. However, there was a
non-significant trend towards an increase in the immune response in
animals treated with the anti-NGF monoclonal antibody.
[0170] By contrast, FIG. 11 shows that following immunization with
chicken gamma-globulin, treatment with anti-NGF produces a
non-significant trend toward a decrease in the immune response.
Animals were immunized with 10 g of chicken gamma-globulin on day a
and treated with-anti-NGF antibody 911 or a control, isotype
matched antibody (10 mg/kg IP) on day 40. On day 42 animals were
boosted with gamma-globulin and on day 47 serum samples were taken
and analyzed by ELISA. There was no significant difference
(p>0.05) in the immune response between the two groups. However
there was a trend towards a decrease in the immune response
following treatment with anti-NGF monoclonal antibody 911.
Example 4
Effect of Anti-NGF Monoclonal Antibodies on Hyperalgesia
[0171] The effect of anti-NGF antibodies on NGF induced thermal
hyperalgesia was investigated. Briefly, adult Fischer female rats
were trained for two sessions a day with the Hargreaves' test for
at least two days prior to treatment. After familiarization with
the apparatus and protocol, animals were randomly assigned to the
control or experimental group. Both groups were tested for baseline
responsiveness and then given intraplantar injections in one paw in
a volume of 50 I under isofluorane anaesthesia. The injections in
each group contained 1.25% carrageenan, in combination with either
90 g of anti-NGF antibody or an indifferent, isotype matched
control antibody.
[0172] Thermal withdrawal latencies were measured at 0, 2, 4, 6 and
24 hours post injection by the Hargreaves' test. As shown in FIG.
12, at 4 and 6 hours after treatment the thermal hyperalgesia that
follows carageenan injection was significantly decreased in animals
treated with anti-NGF monoclonal antibody 911 compared to control
animals.
Example 5
Effect of Anti-NGF Monoclonal Antibodies on Response to
Allergens
[0173] Male C57/BL6 mice (Jackson Laboratories) were treated with
either monoclonal anti-NGF antibody 911 (n=16) or an isotype
matched anti-gD control antibody (clone 1766; n=16). On day-1,
animals were treated with 20 mg/kg of antibody and on days 6, 13,
20 and 22 they were treated with 10 mg/kg of antibody. All antibody
treatments were subcutaneously injected into the scruff of the
neck.
[0174] On days 0 and 14 half of the mice in each group were
sensitized (SN) by intraperitoneal injection of 30 AU of dust mite
antigen (DMA; diluted with Dulbecco's PBS and then 1:2 with ALUM,
as an adjuvant, for a final concentration of 300 AU/ml). The
non-sensitized mice (NS) received an equal volume of Dulbecco's PBS
diluted 1:2 with ALUM as a control.
[0175] Mice were then challenged with inhaled dust mite antigen
(DMA) on days 21 and 22. Dust mite was diluted to 6000 AU/ml using
Dulbecco's PBS plus 0.01% Tween-20 for aerosolizaton. All
inhalation challenges were administered in a Plexiglas pie exposure
chamber. DMA was aerosolized using a PARI IS-2 nebulizer driven at
22 PSI. The nebulizer was filled with 3 ml and run to completion
(30 min.). Total deposited dose/exposure into the lung was
.about.6.5 AU DMA.
[0176] Mice were assessed for airway hyperreactivity, cellular
infiltration into the broncheolar lavage (BAL) fluid, cytokine
levels in BAL, and serum titer against dust mite as well as serum
IgE levels. Briefly, on day 24, 48 hours after the last challenge,
mice were anesthetized, catheterized in the jugular vein and
tracheotomized. Miche were then paralyzed with 0.28 mg/kg
pancuronium and loaded into a Plexiglas flow plethysmograph for
measurement of thoracic expansion and airway pressure. Mice were
ventilated using 100% oxygen at a frequency of 170 bpm and Vt equal
to 9 l/g. Breathing mechanics (lung resistance and dynamic
compliance) were continuously monitored using the Buxco XA data
acquisition program. Mice were given a volume history (5 breaths at
2.5.times.Vt), allowed to stabilize for 2 min before baseline
measurement, and then given a one-time 5 second dose of the agonist
at a body weight adjusted flow rate using a Harvard syringe pump
and syringe application software.
[0177] Blood (serum), BAL and lungs were collected. Serum was
assayed for total and specific IgE and IgG. BAL was obtained by
injecting the same aliquot of NaCI 3 times and was assayed for
total IgE by ELISA. Total white blood cells and cell differentials
were obtained from BAL cells.
[0178] Treatment with anti-NGF monoclonal antibody caused a
significant drop in airway hyperreactivity (FIG. 13), as well as in
inflammation as measured by cellular infiltration into the BAL
(FIG. 14). However, there is still a very high proportion of
eosinophils in BAL from anti-NGF treated animals (FIG. 15).
Anti-NGF antibody treatment also decreased the level of the Th2
cytokine IL-13 in the BAL (FIG. 16).
[0179] Despite its ability to decrease the inflammatory response to
allergen, anti-NGF antibody treatment did not decrease the humoral
immune response to dust mite, either as measured by the total serum
immunoglobulin titer to dust mite (FIG. 17) or by the serum level
of IgE (FIG. 18). This indicates that the antibody did block the
biological effect of NGF, but did not affect the survival or
function of B-lymphocytes.
[0180] To confirm that the anti-NGF antibody 911 was having a
functionally significant effect on NGF, a parallel experiment was
carried out in which mice were injected subcutaneously in the
scruff of the neck with 0.1, 1 or 10 mg/kg of anti-NGF monoclonal
antibody 911 on days 1 and 8, or with NGF at 0.1, 1 or 10 mg/kg on
days 1, 3, 6 and 8. Animals were sacrificed on day 0.9 and examined
for the level of the neuropeptide CGRP in the trigeminal ganglion.
Treatment with 1 or 10 mg/kg of NGF caused an increase in CGRP,
verifying that this peptide is regulated by NGF levels (FIG. 19).
Treatment with 1 or 10 mg/kg of anti-NGF monoclonal antibody 911
caused a decrease in the CGRP content of the ganglion (FIG. 19),
verifying that this dose produces a functionally significant
blockade of endogenous NGF at the time when the immune response was
occurring in the experiment described above.
* * * * *